Abstract

In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca2+.

Immunoblots showing CFTR and KCa3.1 proteins extracted from CFBE bronchial cells expressing wt-CFTR (A, B) and F508del-CFTR (C, D). Membranes were blotted with anti-CFTR (mAb 596 from CFFT, 1:1000, A, C) and anti-KCa3.1 (Alomone, 1:300, B, D) antibodies. Endogenous expression of CFTR and KCa3.1 proteins in the CFBE-wt and CFBE-ΔF508 cell lysates are presented in lane “Total Lysate”. Immunoprecipitation of endogenous CFTR using anti-CFTR antibody followed by co-immunoprecipitation of KCa3.1 is illustrated in lane IP CFTR (B, D), while immunoprecipitation of endogenous KCa3.1 (using anti-KCa3.1 antibody) followed by co-immunoprecitation of CFTR is shown in lane IP KCa3.1 (A, C). Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with CFTR and KCa3.1 antibodies, respectively.

CFTR and KCa3.1 expression in cell lysates and streptavidin pulldowns after cell-surface biotinylation.

T-Rex HEK cells expressing WT-CFTR were transfected with HA-tagged KCa3.1 channels and CFTR expression induced by tetracycline (Tet). Lanes 1 and 4 show CFTR, Na+/K+-ATPase and KCa3.1-HA proteins in the lysate and pulldown (PD) after biotinylation. As controls, lanes 2 and 5 show Na+/K+-ATPAse and KCa3.1-HA proteins in the lysate and pulldown after biotinylation in absence of forskolin stimulation, and lanes 3 and 6 show CFTR in the lysate and pulldown after biotinylation without forskolin stimulation and KCa3.1 transfection. The molecular mass in kDa is indicated.

The temporal autocorrelations and the cross-correlation functions of EGFP-CFTR and KCa3.1-dsRed.

A: Examples of confocal microscopy images obtained after excitation of EGFP-CFTR and KCa3.1-dsRed at 488 nm and 561 nm respectively, in low Ca2+ conditions. Merged figures support colocalization of EGFP-CFTR and KCa3.1-dsRed at the membrane (orange). B: Examples of confocal microscopy images obtained for EGFP-CFTR and KCa3.1-dsRed following CPA pretreatment in zero Ca2+ and addition of external Ca2+ to initiate Ca2+ influx. Merged figures (orange) showed the formation of larger KCa3.1 clusters after internal Ca2+ increase. C: Evidence for CFTR/KCa3.1 interactions provided by cross-correlation measurements (see in Materials and Methods section). CFTR-KCa3.1 interactions on the plasma membrane yielded a non-zero cross-correlation function (black) at the slow time scales of the correlation function. This strongly suggests that only the slow populations of CFTR and KCa3.1 interact.

A: CFTR and KCa3.1 number densities (molecule/μm2) under control and Ca2+ influx conditions. KCa3.1 number density dramatically decreases (4-fold) during Ca2+ influx induced by pretreatment with CPA (cyclopiazonic acid) followed by exposure to extracellular Ca2+, consistent with either internalization of this channel or its clustering. B: CFTR and KCa3.1 degree of aggregation (DA) under control and Ca2+ influx conditions. KCa3.1 degree of aggregation increases significantly (4-fold) during Ca2+ influx. The 4-fold decrease in number density is accounted for by the 4-fold increase in KCa3.1 cluster size (DA). C: Significant increase in the fraction of KCa3.1 interacting with CFTR in response to a rise in intracellular Ca2+ concentration. Protein/protein interactions occurred on a slow time scale (D) and most interactions involved molecules that were immobilized on the plasma membrane (E). Statistical analysis based on n = 62 cells for control experiments and n = 21 cells for measurements in CPA+ Ca2+ conditions.

Schematic representation of the effect of internal Ca2+ on KCa3.1 interactions and dynamics.

Illustration of the increase in CFTR/KCa3.1 clustering in response to an internal Ca2+ rise. PM refers to plasma membrane. This scheme accounts for the decrease in KCa3.1 density in the presence of Ca2+ as most KCa3.1 channels form aggregates with CFTR.